猪CD163基因的单碱基编辑研究

doi:10.11843/j.issn.0366-6964.2022.04.005

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (4): 1041-1050.doi: 10.11843/j.issn.0366-6964.2022.04.005

猪CD163基因的单碱基编辑研究

赵为民^1,2, 王慧利^1,2, 曹少先^1,2, 郭日红^1,2, 王泽平^1,3, 陈哲^1,2, 徐奎⁴, 付言峰^1,2, 李碧侠^1,2, 任守文^1,2, 程金花^1,2*

1. 江苏省农业科学院畜牧研究所江苏省农业种质资源保护与利用平台, 南京 210014;
2. 农业农村部种养结合重点实验室, 南京 210014;
3. 江苏省农业科学院宿迁农科所, 宿迁 223800;
4. 中国农业科学院深圳农业基因组研究所, 深圳 518124

收稿日期:2021-08-05 出版日期:2022-04-23 发布日期:2022-04-25
通讯作者: 程金花,主要从事猪的育种与生产研究,E-mail:jhcheng@jaas.ac.cn
作者简介:赵为民(1983-),男,湖北钟祥人,博士,副研究员,主要从事猪的抗病育种研究,E-mail:zhao_weimin1983@aliyun.com
基金资助:
财政部和农业农村部：国家现代农业产业技术体系资助；江苏省苏北科技专项(XZ-SZ201921)；扬州市重点研发项目(YZ2021037)；江苏省现代农业后补助项目(BE2020358)；江苏省自主创新资金项目(CX (19)2016)

The Study of Base Editing of Porcine CD163 Gene

ZHAO Weimin^1,2, WANG Huili^1,2, CAO Shaoxian^1,2, GUO Rihong^1,2, WANG Zeping^1,3, CHEN Zhe^1,2, XU Kui⁴, FU Yanfeng^1,2, LI Bixia^1,2, REN Shouwen^1,2, CHENG Jinhua^1,2*

1. Jiangsu Germplasm Resources Protection and Utilization Platform, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
2. Key Laboratory for Crop and Animal Integrated Farming of Ministry of Agriculture and Rural Affairs, Nanjing 210014, China;
3. Suqian Institute of Agricultural Sciences, Jiangsu Academy of Agricultural Sciences, Suqian 223800, China;
4. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518124, China

Received:2021-08-05 Online:2022-04-23 Published:2022-04-25

摘要/Abstract

摘要： 旨在利用YE1-BE3-FNLS载体对猪的CD163基因进行C>T的单碱基突变，形成终止密码子TAA，从而导致CD163基因翻译的提前终止。利用网站在猪CD163基因的第7外显子筛选到高效率的sgRNA序列使其符合C5位点，并且C5后续的两个碱基为AA，CAA与起始密码子ATG之间的碱基数为3的整数倍。PCR扩增CD163-sgRNA和YE1-BE3-FNLS载体中的APOBEC-Cas9n-UGI片段，通过体外转录形成CD163-sgRNA和APOBEC-Cas9n-UGI mRNA。体外培养猪卵母细胞并进行孤雌激活，利用显微操作仪对孤雌胚胎进行CD163-sgRNA和APOBEC-Cas9n-UGI mRNA注射，待胚胎发育至桑椹胚和囊胚阶段，提取胚胎基因组DNA对单碱基编辑区域进行PCR扩增测序。结果显示，在49个候选sgRNA中成功筛选出一个CD163-sgRNA，利用PCR成功扩增了CD163-sgRNA和APOBEC-Cas9n-UGI片段，注射CD163-sgRNA和APOBEC-Cas9n-UGI mRNA后的猪孤雌胚胎发育到2~4细胞期。挑选注射的20个2~4细胞期胚胎经过PCR扩增与产物测序后发现有12个胚胎的CD163基因的第7外显子的预期位点C (num1479)>T，单碱基编辑效率约60%。对CD163-sgRNA的off-target分析发现，在错配3个碱基的情况下该CD163-sgRNA有5个off-target区域，对这5个区域进行PCR扩增和sanger测序分析后发现都没有发生C>T的突变。本研究利用YE1-BE3-FNLS工具在胚胎水平上对猪的CD163基因进行了单碱基编辑，成功使得该基因第7外显子预期位点(num1479) C变为T，从而使得密码子CAA变为TAA，可提前终止CD163基因的翻译。

关键词: CRISPR/Cas9, 单碱基编辑, CD163基因, 猪

Abstract: The aim of this study was to use the YE1-BE3-FNLS vector to carry out a single-base mutation of C>T in the porcine CD163 gene to form the stop codon TAA, which would lead to the premature termination of the translation of the CD163 gene. The website was used to design high-efficiency sgRNA sequence at the 7th exon of porcine CD163 gene and make it conform to C5 feature, and the following two bases of C5 were AA, and the number of bases between CAA and the start codon ATG was the integer multiple of 3. The CD163-sgRNA and APOBEC-Cas9n-UGI fragment in the YE1-BE3-FNLS vector was amplified by PCR, and CD163-sgRNA and APOBEC-Cas9n-UGI mRNA were produced by in vitro transcription. Porcine oocytes were cultured in vitro and undergone parthenogenetic activation. The CD163-sgRNA and APOBEC-Cas9n-UGI mRNA were injected into parthenogenetic embryos by micromanipulator. After the embryo developed to the stage of morula and blastocyst, the genomic DNA of the embryo was extracted and its single-base editing region was subjected to PCR amplification and sequencing. The results showed that a CD163-sgRNA was successfully screened among 49 candidate sgRNAs. The CD163-sgRNA and APOBEC-Cas9n-UGI fragments were successfully amplified by PCR, and the porcine parthenogenetic embryos developed to the 2-4 cell stage after injection of CD163-sgRNA and APOBEC-Cas9n-UGI mRNA. After PCR amplification and product sequencing of selected injected 20 embryos at 2-4 cell stage, there were 12 embryos with the expected site C(num1479)>T of the 7th exon of the CD163 gene, and the single-base editing efficiency was about 60%. It was found that the CD163-sgRNA had 5 off-target regions in the case of a mismatch of 3 bases based on the off-target analysis. After analyzing these 5 regions, it was found that there was no C>T mutation using PCR amplification and sanger sequencing. The pig CD163 gene was suffered with base editing by YE1-BE3-FNLS tool at the embryo level in this study, and the 7th exon expected site (num1479) C was successfully changed to T, which can terminate the translation of CD163 gene in advance by making the codon CAA to TAA.

Key words: CRISPR/Cas9, base editing, CD163 gene, pig

中图分类号:

S828.2

赵为民, 王慧利, 曹少先, 郭日红, 王泽平, 陈哲, 徐奎, 付言峰, 李碧侠, 任守文, 程金花. 猪CD163基因的单碱基编辑研究[J]. 畜牧兽医学报, 2022, 53(4): 1041-1050.

ZHAO Weimin, WANG Huili, CAO Shaoxian, GUO Rihong, WANG Zeping, CHEN Zhe, XU Kui, FU Yanfeng, LI Bixia, REN Shouwen, CHENG Jinhua. The Study of Base Editing of Porcine CD163 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1041-1050.

参考文献 36

[1]	BARRANGOU R,FREMAUX C,DEVEAU H,et al.CRISPR provides acquired resistance against viruses in prokaryotes[J].Science, 2007,315(5819):1709-1712.
[2]	HORVATH P,BARRANGOU R.CRISPR/Cas,the immune system of bacteria and archaea[J].Science, 2010,327(5962):167-170.
[3]	JINEK M,CHYLINSKI K,FONFARA I,et al.A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J].Science, 2012,337(6096):816-821.
[4]	MALI P,YANG L H,ESVELT K M,et al.RNA-guided human genome engineering via Cas9[J].Science, 2013,339(6121):823-826.
[5]	HSU P D,LANDER E S,ZHANG F.Development and applications of CRISPR-Cas9 for genome engineering[J]. Cell, 2014,157(6):1262-1278.
[6]	HILTON I B,GERSBACH C A.Enabling functional genomics with genome engineering[J].Genome Res, 2015,25(10):1442-1455.
[7]	JIANG W Z,ZHOU H B,BI H H,et al.Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice[J].Nucleic Acids Res, 2013,41(20):e188.
[8]	RUAN J X,XU J,CHEN-TSAI R Y,et al.Genome editing in livestock:are we ready for a revolution in animal breeding industry?[J].Transgenic Res, 2017,26(6):715-726.
[9]	RAZZAQ A,SALEEM F,KANWAL M,et al.Modern trends in plant genome editing:an inclusive review of the CRISPR/ Cas9 toolbox[J].Int J Mol Sci, 2019,20(16):4045.
[10]	TRAN N T,SOMMERMANN T,GRAF R,et al.Efficient CRISPR/Cas9-mediated gene Knockin in mouse hematopoietic stem and progenitor cells[J].Cell Rep, 2019,28(13):3510-3522.e5.
[11]	ZHAO H,LI Y,HE L J,et al.In vivo AAV-CRISPR/Cas9-mediated gene editing ameliorates atherosclerosis in familial hypercholesterolemia[J].Circulation, 2020,141(1):67-79.
[12]	KOMOR A C,KIM Y B,PACKER M S,et al.Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage[J].Nature, 2016,533(7603):420-424.
[13]	GAUDELLI N M,KOMOR A C,REES HA，et al.Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage[J].Nature, 2017,551(7681):464-471.
[14]	ZUO E W,SUN Y D,WEI W，et al.Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos[J].Science, 2019,364,364(6437):289-292.
[15]	JIN S A,ZONG Y,GAO Q,et al.Cytosine,but not adenine,base editors induce genome-wide off-target mutations in rice[J].Science, 2019,364(6437):292-295.
[16]	GRÜNEWALD J,ZHOU R H,GARCIA S P,et al.Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors[J].Nature, 2019,569(7756):433-437.
[17]	ZHOU C Y,SUN Y D,YAN R,et al.Off-target RNA mutation induced by DNA base editing and its elimination by mutagenesis[J].Nature, 2019,571(7764):275-278.
[18]	ZUO E W,SUN Y D,YUAN T L,et al.A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects[J].Nat Methods, 2020,17(6):600-604.
[19]	DOMAN J L,RAGURAM A,NEWBY G A,et al.Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors[J].Nat Biotechnol, 2020,38(5):620-628.
[20]	TIAN K G,YU X L,ZHAO T Z,et al.Emergence of fatal PRRSV variants:unparalleled outbreaks of atypical PRRS in China and molecular dissection of the unique hallmark[J].PLoS One, 2007,2(6):e526.
[21]	CALVERT J G,SLADE D E,SHIELDS S L,et al.CD163 expression confers susceptibility to porcine reproductive and respiratory syndrome viruses[J].J Virol, 2007,81(14):7371-7379.
[22]	VAN GORP H,DELPUTTE P L,NAUWYNCK H J.Scavenger receptor CD163,a Jack-of-all-trades and potential target for cell-directed therapy[J].Mol Immunol, 2010,47(7-8):1650-1660.
[23]	STOIAN A M M,ROWLAND R R R.Challenges for Porcine Reproductive and Respiratory Syndrome (PRRS) vaccine design:reviewing virus glycoprotein interactions with CD163 and targets of virus neutralization[J].Vet Sci, 2019,6(1):9.
[24]	HUANG C,BERNARD D,ZHU J Q,et al.Small molecules block the interaction between porcine reproductive and respiratory syndrome virus and CD163 receptor and the infection of pig cells[J].Virol J, 2020,17(1):116.
[25]	WANG H T,SHEN L C,CHEN J Y,et al.Deletion of CD163 exon 7 confers resistance to highly pathogenic porcine reproductive and respiratory viruses on pigs[J].Int J Biol Sci, 2019,15(9):1993-2005.
[26]	BURKARD C,LILLICO S G,REID E,et al.Precision engineering for PRRSV resistance in pigs:macrophages from genome edited pigs lacking CD163 SRCR5 domain are fully resistant to both PRRSV genotypes while maintaining biological function[J].PLoS Pathog, 2017,13(2):e1006206.
[27]	YANG H Q,ZHANG J,ZHANG X W,et al.CD163 knockout pigs are fully resistant to highly pathogenic porcine reproductive and respiratory syndrome virus[J].Antiviral Res, 2018,151:63-70.
[28]	BURKARD C,OPRIESSNIG T,MILEHAM A J,et al.Pigs lacking the scavenger receptor cysteine-rich domain 5 of CD163 are resistant to porcine reproductive and respiratory syndrome virus 1 infection[J].J Virol, 2018,92(16):e00415-18.
[29]	GUO C H,WANG M,ZHU Z B,et al.Highly efficient generation of pigs harboring a partial deletion of the CD163 SRCR5 domain,which are fully resistant to porcine reproductive and respiratory syndrome virus 2 infection[J].Front Immunol, 2019,10:1846.
[30]	MONTAGUE T G,CRUZ J M,GAGNON J A,et al.CHOPCHOP:a CRISPR/Cas9 and TALEN web tool for genome editing[J].Nucleic Acids Res, 2014,42(W1):W401-W407.
[31]	OLADZAD A,PORCH T,ROSAS J C,et al.Single and multi-trait GWAS identify genetic factors associated with production traits in common bean under abiotic stress environments[J].G3 (Bethesda), 2019,9(6):1881-1892.
[32]	THURONYI B W,KOBLAN L W,LEVY J M,et al.Continuous evolution of base editors with expanded target compatibility and improved activity[J].Nat Biotechnol, 2019，37(9):1070-1079.
[33]	FU Y F,FODEN J A,KHAYTER C,et al.High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells[J].Nat Biotechnol, 2013,31(9):822-826.
[34]	GRAVERSEN J H,MADSEN M,MOESTRUP S K.CD163:a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma[J].Int J Biochem Cell Biol, 2002,34(4):309-314.
[35]	XU K,ZHOU Y R,MU Y L,et al.CD163 and pAPN double-knockout pigs are resistant to PRRSV and TGEV and exhibit decreased susceptibility to PDCoV while maintaining normal production performance[J].elife, 2020,9:e57132.
[36]	姚旭东,蒙亚琦,任秀美奥,等.利用单碱基编辑系统定点编辑哈萨克羊MSTN 基因的研究[J].畜牧兽医学报,2021,52(8):2162-2170.YAO X D,MENG Y Q,REN X M A,et al.Study of Kazakh sheep MSTN gene site-directed editing based on the base editing system[J].Acta Veterinaria et Zootechnica Sinica, 2021,52(8):2162-2170.

猪CD163基因的单碱基编辑研究

The Study of Base Editing of Porcine CD163 Gene

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 36

相关文章 15

编辑推荐

Metrics

本文评价

[1]	康佳威, 黄宣凯, 王志鹏, 张爱珍, 孟芳荣, 盖鹏, 包军付, 孙可心, 宋少康, 孙攀, 陈一川, 张蕾, 高圣玥, 常铭航. 大白猪生长、繁殖和体尺性状遗传参数估计[J]. 畜牧兽医学报, 2024, 55(5): 1936-1944.
[2]	孙雯莉, 王浩奇, 泽里磋, 高雨樊, 张非凡, 张健, 段梦琪, 商鹏, 强巴央宗. 藏猪促炎因子(IL-1β、IL-6、TNF-α)多态性及其表达与免疫性状的关联分析[J]. 畜牧兽医学报, 2024, 55(5): 1958-1969.
[3]	李婉君, 徐皆欢, 何孟纤, 孔钰婷, 张德福, 戴建军. 细胞松弛素B改善冷冻引起的猪卵母细胞皮质颗粒迁移障碍[J]. 畜牧兽医学报, 2024, 55(5): 1999-2010.
[4]	韩阳, 关帅印, 李振, 周赛赛, 袁红根, 宋云峰. 猪圆环病毒3型Rep蛋白的原核表达及酶活性分析[J]. 畜牧兽医学报, 2024, 55(5): 2061-2071.
[5]	宋晓晴, 邓瑞德, 李欣, 李姣, 李润成, 杜丽飞, 董伟, 葛猛. PCV4 Cap抗体ELISA检测方法的建立及血清流行病学调查[J]. 畜牧兽医学报, 2024, 55(5): 2072-2079.
[6]	周扬, 吴炜姿, 曹伟胜, 王福广, 许秀琼, 钟文霞, 吴立炀, 叶健, 卢受昇. 基于Nanopore测序技术的非洲猪瘟病毒全基因组测序方法建立[J]. 畜牧兽医学报, 2024, 55(5): 2080-2089.
[7]	马茹梦, 赵玉梁, 马明爽, 国桂海, 刘芯孜, 李佳璇, 崔文, 姜艳平, 单智夫, 周晗, 王丽, 乔薪瑗, 唐丽杰, 王晓娜, 李一经. 不同猪源受体菌表达猪流行性腹泻病毒保护性抗原S1诱导免疫应答的比较研究[J]. 畜牧兽医学报, 2024, 55(5): 2090-2099.
[8]	徐红, 商红旗, 张雪, 钱嘉莉, 王传红, 宋旭, 宝梅英, 刘诗雨, 张格格, 郭容利, 赵永祥, 范宝超, 李彬. C8orf4基因编码蛋白对猪流行性腹泻病毒体外复制的抑制效应[J]. 畜牧兽医学报, 2024, 55(5): 2100-2108.
[9]	王静, 张淑娟, 胡霞, 刘向阳, 张兴翠, 宋振辉. CD44通过影响猪流行性腹泻病毒复制调节钠氢交换体3活性[J]. 畜牧兽医学报, 2024, 55(5): 2176-2185.
[10]	王吉英, 尹蕊如, 谢星, 王海燕, 刘胡栋, 胡辉, 熊祺琰, 冯志新, 邵国青, 于岩飞. 猪肺炎支原体乳酸脱氢酶在诱导猪支气管上皮细胞凋亡中的作用[J]. 畜牧兽医学报, 2024, 55(5): 2195-2205.
[11]	胡泽奇, 李润成, 谭祖明, 谢秀艳, 王江平, 秦乐娟, 李荣, 葛猛. PEDV、PoRVA和PDCoV TaqMan三重RT-qPCR检测方法的建立与初步应用[J]. 畜牧兽医学报, 2024, 55(5): 2267-2272.
[12]	邱梅玉, 张雪梅, 张宁, 刘明军. 引导编辑技术的研究进展及应用[J]. 畜牧兽医学报, 2024, 55(4): 1345-1355.
[13]	彭佩雅, 陈钰焓, 杨龙, 王铭, 赵芮葶, 何俊, 印遇龙, 刘梅. 家畜基因组拷贝数变异研究进展[J]. 畜牧兽医学报, 2024, 55(4): 1356-1369.
[14]	黄杰, 阮子豪, 蔡瑞. 抗菌肽在猪精液常温保存中的应用研究进展[J]. 畜牧兽医学报, 2024, 55(4): 1401-1411.
[15]	徐俊杰, 张璐通, 王津洁, 陈晓晨, 何伟先, 蔡传江, 褚瑰燕, 杨公社. 基于多组学与网络药理学探究淫羊藿对后备母猪发情的作用[J]. 畜牧兽医学报, 2024, 55(4): 1615-1628.